A pilot study on post-thawing quality of Iberian red deer spermatozoa (epididymal and electroejaculated) depending on glycerol concentration and extender osmolality

Abstract

The optimization of cryopreservation extenders is a fundamental issue for adequately performing germplasm banking on wild species. We have tested two glycerol concentrations (4 and 8%), and three extender osmolalities (320, 380 and 430 mOsm/kg; before adding cryoprotectants), for cryopreservation of epididymal and ejaculated sperm samples from Iberian red deer. All the extenders were based on Tes-Tris and fructose (for osmolality adjustment), and complemented with 20% egg yolk. Epididymal and ejaculated sperm samples were obtained from the cauda epididymis (post-mortem) and using electroejaculation, respectively. Samples were diluted 1:1 with each extender and equilibrated for 2 h at 5 degrees C. Then, they were diluted down to 100x10(6) sperm/mL and frozen at -20 degrees C/min. Post-thawed samples were assessed for motility (CASA), HOS test, proportion of swollen (osmotically challenged) cells in the untreated sample, viability and acrosomal status. For epididymal samples, 8% glycerol rendered a slightly higher proportion of intact acrosomes on viable spermatozoa than 4%; regarding extender osmolality, 380 and 430 mOsm/kg rendered higher motility results, and the 430 mOsm/kg yielded the lowest proportion of swollen spermatozoa. For ejaculated samples, 4% glycerol yielded more viable spermatozoa than 8%; for extender osmolality, 320 mOsm/kg rendered the highest percentages of progressively motile and viable spermatozoa, although 380 mOsm/kg extender was not significantly different. These results show that sample source influences extender suitability, and that extenders should be isoosmotic or rather slightly hyperosmotic. Future studies should test multiple glycerol concentrations and extender osmolalities in order to adjust them to these kinds of sample.

The Application of Reproductive Technologies to Natural Populations of Red Deer

“…In our experiments with Iberian red deer, we used a 250 × 30 mm probe with three longitudinal electrodes (Martínez et al. 2004; Martínez‐Pastor et al. 2004, 2006b), achieving ejaculation at average voltage and amperage values of 4.5 V and 90 mAmp, respectively.…”

“…2000; Anel et al. 2002; Martínez‐Pastor et al. 2002, 2006b) or by retrograde flushing of the cauda from the vas deferens (Garde et al.…”

“…Thus, CASA systems have been used to analyse sperm motility parameters (Malo et al. 2005b; Martínez‐Pastor et al. 2005c, 2006b,c), and it has been possible to identify subpopulations defined by motility patterns, and their changes in different circumstances (Martínez‐Pastor et al.…”

“…Moreover, fluorescent probes (either by fluorescence microscopy or by flow cytometry) have been used to analyse different physiological parameters of red deer semen, as viability and acrosomal status (Anel et al. 2002; Martínez‐Pastor et al. 2006a–c), or mitochondrial membrane potential (Martínez‐Pastor et al.…”

Improving the effect of incubation and oxidative stress on thawed spermatozoa from red deer by using different antioxidant treatments

“…Cryopreservation of epididymal spermatozoa is feasible and practical for the creation of germplasm banks for wild deer species (Garde et al 2006;Martínez-Pastor et al 2006b). Nevertheless, conditions must be optimised for the successful post-thawing application of such samples.…”